Bradykinin is a vasoactive peptide released from the precursor plasma kininogens by kallikrein and other enzymes (Silva et al., Amer. J. Physiol. 156: 261-274 (1949)). Bradykinin has been described to have multiple physiologic functions, including the stimulation of prostacyclin production (Hong, S. L., Thromb. Res. 18, 787 (1980); Crutchley et al., Biochim Biophy Acta 751, 99 (1983)) and the stimulation of the release of plasminogen activators (Smith et al., Blood 66, 835 (1983)). Bradykinin induces superoxide formation and endothelium-dependent smooth muscle hyperpolarization (Holland, J. A. et al., J. Cell Physiol. 143, 21 (1990); Nakashima, M. et al., J. Clin. Invest. 92, 2867 (1993)). Along with acetylcholine, bradykinin is the major inducer of nitric oxide formation (Palmer, R. M. J. et al., Nature 327, 524 (1987)). Bradykinin has been characterized to produce vasodilation in most vascular beds which in the coronary artery circulation results in increased blood flow (Line et al., J. Mol. Cell Cardiol. 24, 909 (1992)). These latter features have led some to characterize bradykinin as a cardioprotective agent (Line et al., supra; Gohlke et al., Hypertension 23, 411 (1994); Parratt et al., Cardiovascular Research 28, 183 (1994); Zanzinger et al., Cardiovascular Research 28, 209 (1994)). Bradykinin's elevation by angiotensin converting enzyme inhibitors is believed to be the mechanism by which these drugs promote their beneficial effects on heart failure.
In addition to the delivery of bradykinin, its parent proteins, high (HK) and low (LK) molecular weight kininogens, also have the ability to be selective inhibitors of α-thrombin, inhibiting α-thrombin's ability to activate cells without interfering with its enzymatic ability (Meloni et al., J. Biol. Chem. 266, 6786 (1991); Puri et al., Blood 77, 500 (1991)). This activity was believed to be a unique function for the kininogens; one which had not been ascribed to other proteins. Most naturally occurring human protein inhibitors of α-thrombin are directed towards its protease activity. HK and LK are selective inhibitors of thrombin's ability to activate platelets by blocking α-thrombin from binding to the platelet membrane (Meloni et al., supra; Puri et al., supra). This activity of the kininogens appeared to be localized to domain 3 on their heavy chain since isolated domain 3 retains that activity (Jiang et al., J. Biol. Chem. 267, 3712 (1992)).
Inhibition of platelet activation by domain 3 is observed by a marked decrease in the platelet's ability to aggregate and secrete their granule contents. The granule contents comprise proteins which participate in hemostasis, thrombosis, and the inflammatory response. Inhibition of endothelial cell activation may similarly be observed by a decrease in secretion of endothelial cell contents such as tissue plasminogen activator and von Willebrand factor.
The isolated domain 3 from a tryptic digest of LK, like its parent proteins HK and LK, functions to inhibit cell activation by blocking thrombin binding to its target cells. This polypeptide is a selective inhibitor of thrombin-induced platelet activation. Administration of domain 3 in vitro therefore does not impact on induction of platelet activation by physiological substances other than thrombin, such as, for example collagen, adenosine diphosphate, epinephrine and platelet activating factor.
Interventional procedures for coronary artery disease such as coronary thrombolysis or percutaneous transluminal coronary angioplasty have reduced mortality from acute coronary thrombosis. However, after intracoronary thrombolysis with lytic agents, the reocclusion rate is high. The major cause for reocclusion is platelet thrombus. When artificial dacron grafts are anastomosed to human arteries, up to 30% of all patients will develop a platelet thrombosis within hours of surgery. This expected high complication rate frequently requires an additional operation with attendant complications. Thus, additional therapies are needed to prevent these reocclusion events due to platelet thrombi.
Two competing classes of antiplatelet agents for the prevention of coronary thrombosis are being considered. One class of agents, including monoclonal antibody 7E3, aims to block the final common pathway of platelet activation by inhibiting glycoprotein IIb/IIIa (GPIIb/IIIa), integrin αIIbβ3. 7E3 is an effective agent, but it is a murine antibody and is antigenic in humans. A second class of antiplatelet agents inhibit a presumed, primary initiating agent of platelet activation, α-thrombin. Infusions of Phe-Pro-Arg-chloromethylketone (PPACK), a potent inhibitor of α-thrombin's proteolytic activity, prolongs the bleeding time, a crude measure of platelet function (Hanson, S. R. et al., Proc. Natl. Acad. Sci. 85, 3184-3188 (1988)). The first generation of potent α-thrombin proteolytic inhibitors to enter into clinical trials is a recombinant product derived from medicinal leeches, hirudin. This compound, which is a small molecular mass and is not considered to be antigenic, is a potent anti-thrombin. A synthetic analog of hirudin, hirulog, combines the anion exosite I binding properties of hirudin with the proteolytic inhibitory activity of PPACK. In Phase III clinical trials, both drugs were effective inhibitors of platelet activation. The tradeoff for effective anticoagulation, however, was increased hemorrhage into brain leading to the termination of three clinical trials. These non-selective inhibitors of α-thrombin have an antithrombotic efficiency dose close to their toxicity dose and are not clinically tolerated and, thus, may never have commercial significance.
An ideal anti-thrombotic to prevent arterial thrombosis would be one which prevents platelet and endothelial cell activation without preventing the proteolytic activity of α-thrombin to clot fibrinogen and activate protein C, factor XIII, and factors V and VIII. Only two known proteins, high molecular weight (HK) and low molecular weight (LK) kininogens, are naturally occurring selective anti-thrombins (Meloni, F. J. et al., J. Biol. Chem. 266; 6786-6794 (1991); Puri, R. N. et al., Blood 77:500-507 (1991)). Both low and high molecular weight kininogens have identical amino acid sequences from their amino-terminus through 12 amino acids beyond the carboxy-terminus of bradykinin. LK and HK share a common heavy chain (62 kDa), the bradykinin (BK) moiety (0.9 kDa), and the first 12 amino acids of the amino terminal portion of each of their “light chains” (Takagaki, Y. et al., J. Biol. Chem. 260:8601-8609 (1985); Kitamura, N. et al., J. Biol. Chem., 260:8610-8617 (1985)). This identity corresponds to residues 1 through about residue 383. See Salveson et al., Biochem J. 234, 429 (1986); Kellerman et al., Eur. J. Biochem. 154, 471 (1986). They diverge in the size of their light chains; the light chain of LK is 4 kDa; that of HK is 56 kDa. Takagaki et al., supra; Kitamura et al., supra.
There is a need for improved methods of identification, as well as the identification of new compounds which specifically inhibit thrombin-induced platelet or other cell activation, and for compounds which prevent platelet aggregation.